DESCRIPTION: Orthopoxviruses can replicate productively in a very broad range of cell lines in vitro, but their replications in some specific cell lines are abortive unless the so-called "host-range" genes are intact in their genome [1]. In our preliminary studies, a functional host-range gene, either K1L or C7L or CP77, was found to be also required for vaccinia virus (W) to replicate productively in primary human cells and for W virulence in a mouse model. However, the mechanisms by which these host-range genes support viral growth in otherwise non-permissive host are not clear. K1L, C7L and CP77 share no significant amino acid sequence homology, but both CP77 and K1L encode multiple ankyrin repeats. Because ankyrin repeats proteins typically function in mediating specific protein-protein interactions [2, 3], we hypothesize that CP77 and K1L interact with specific viral or/and cellular factors via their ankyrin repeats to circumvent some intracellular barriers of poxvirus replication. This hypothesis is supported by our preliminary studies that demonstrated: (1) variable surface residues of a few consecutive K1L ankyrin repeats are essential for its host-range function in human cells;(2) K1L interacts with W C10L protein during viral replication; and (3) K1L interacts directly with extracellular signal-regulated kinase 5 (ERK5), a crucial component of mitogen- activated protein kinase (MAPK) cascades regulating multiple cellular processes. We will further test our hypothesis by accomplishing the following specific aims: 1. To explore the molecular basis for the host-range function through structure-function analysis ofK1LandCP77. 2. To determine whether K1L mediates its host-range function through interactions with ERK5 . and WCIOL Accomplishing these two specific aims will provide some fundamental knowledge about the intracellular barriers of orthopoxviral replication and viral strategies of circumventing these barriers. This will benefit the development of safer vaccines and novel antivirals for pathogenic Orthopoxviruses such as variola virus and monkeypox virus. [unreadable] [unreadable] [unreadable]